The B7:CD28/CTLA-4 costimulatory pathway has a critical role in regulating T cell activation, differentiation and tolerance, and is a promising therapeutic target. PD-1 is structurally related to CTLA-4 and has an ITIM motif in its cytoplasmic tail. PD-1 deficient mice develop an autoimmune-like disease. Our recent studies show that the newly discovered B7 homologues, PD-L1 and PD-L2, are ligands for PD-1, and can inhibit T cell proliferation and cytokine production in vitro. The expression of PD-L1 and PD-L2 on nonlymphoid tissues, as well as professional APCs, supports a role for this pathway in regulating peripheral T cell tolerance. The delineation of this pathway has revealed a new means by which T cell responses are regulated, and raised questions about its role in regulating T cell activation and tolerance, and its relationship with the B7:CD28/CTLA-4 pathway. The goals of this project are to investigate the roles of PD-L1 and PD-L2 in regulating T cell activation and tolerance, and interactions between the B7:CD28/CTLA-4 and PD-l:PD-L1/PD-L2 pathways: We will 1) analyze the function of PD-Li and PD-L2 in regulating naive and activated antigen-specific T cells and helper T cell dependent humoral immune responses. We have generated anti-PD-Ll and anti-PD-L2 mAbs and are generating mice lacking PD-L1 and/or PD-L2. These tools provide a definitive means for determining when and how PD-L1 and PD-L2 exert their effects during an immune response, 2) investigate the roles of PD-L1 and PD-L2 in regulating peripheral T cell tolerance. We will use DO.11 TCR Tg T cells to visualize the impact of blockade or elimination of PD-L1 and/or PD-L2 on the responses of antigen-specific CD4+ T cells to immunogenic and tolerogenic stimuli. 3) Analyze the interactions between the PD-1:PD-L1/PD-L2 and B7:CD28/CTLA-4 pathways. We will examine whether these pathways regulate the expression of each other, and evaluate functional interactions. The availability of mice lacking B7-1 and/or B7-2, together with mice lacking CD28 and/or CTLA-4, provide us with unique opportunities to analyze these interactions. These approaches should provide fundamental information about the role of the PD-1:PDL1/PD-L2 pathway in regulating T cell activation and tolerance, and its interactions with the B7:CD28/CTLA-4 pathway. These studies may thereby assist in the design of optimal therapeutic strategies for manipulating the B7:CD28/CTLA-4 pathway and indicate the therapeutic potential of PD-1:PD-L1/PD-L2 pathway manipulation.